4 MECHANICAL VENTILATION OF MICE

Lothar A. Schwarte'12 andel an Ince

J Department ofPhysiology, Academic Medical Center, University ofAmsterdam, Amsterdam the Netherlands 2 Clinic ofAnesthesiology, Heinrich-Heine-University Dusseldorf, Germany

INTRODUCTION

Due to the expanding interest in mouse research (e.g. the emerging field of functional genomics), there is a growing need for (patho-)physiological relevant, stable in vivo murine models, often requiring ventilatory support. Numerous techniques available for in vivo research of larger animals have been miniaturized and adapted, ranging from vascular catheterisation to measurement of cardiac output and organ blood flows. However, a major difficulty in this process of miniaturization is encountered at the level of mechanical ventilation, mainly because of the small size of mice and the technically demanding murine breathing pattern. To summarize the current state of mechanical mouse ventilation, we have updated our recent review on this subject' and have included new data from others and from our own studies on this topic. The following aspects of murine respiratory support will be discussed: Murine respiratory physiology, indications of when to use mechanical ventilation in mice, airway access, types of ventilation of the mouse, and monitoring and side effects of mechanical ventilation.

C. Ince (ed.), The Physiological Genomics of the Critically Ill Mouse © Kluwer Academic Publishers 2004 36 Physiological Genomics of the Critically III Mouse

MURINE RESPIRATORY PHYSIOLOGY

The physiological values for murine respiratory parameters (e.g. "normal" or "standard" values) stated in the literature are still divergent. This is mainly caused by technical difficulties in the measurement of these variables 2, large inter-strain variability 3,4 and differences in experimental conditions (e.g. free moving vs. restrained) 5 Parameters for the spontaneous ventilation of adult mice are given in Table 4-l.Mechanical ventilation has also been accompliched in newborn mice however suitable ventilators and parameters are required6-8 The table for adult mice indicatesd the following characteristics of mice ventilation: RR = 180-270 'min-l, Vt = 0.1-0.2 ml and TiiTt = 0.30-0.35. Only little information is currently available on the lung mechanics of micel5• Murine lung-thorax compliance was measured in pancuronium-paralyzed, mechanically ventilated mice (BALB/c, no weight stated) in the range of 31- 38 f.ll·cm H20-l under control conditions, and 12 f.ll·cm H20-l after experimental surfactant inactivation 16. Respiratory system resistance was measured in age-matched A/J- and C3H/HeJ-mice, revealing significant strain-differences under baseline conditions (1 vs. 2 cm H20/ml/sec) and especially after i.v. acetylcholine-treatment (3 vs. 15 cm H20/mllsec)17. In the same study significant differences were also found for the respiratory

system elastance under both conditions (25 vs. 30 cm H20·ml-l at baseline and 28 vs. 95 cm H20'ml-l after i.v. acetylcholine). Again, one has to realize that marked differences exist between mouse strains in respect to respiratory physiology and pathophysiology. Contradicting data is available on physiologic values for murine arterial blood gasses, regarded as the gold standard for the evaluation of adequacy of ventilation. It has been suggested

that mice have considerably lower alveolar and arterial PC02 than other mammals, as low as 20 mmHg (at pH 7.4)18. Indeed, recent data support these lower PC02 (24 mmHg in chronically instrumented 129SV/J x ICR mice7, 34 mmHg in chronically instrumented C57BLl6 miceI9). Although effects of instrumentation7,19 or restrainment cannot completely be excluded in these studies, the data does indicate a lower PaC02 for the mouse, e.g. compared to mammals ranging from rats to humans (PaC02 33-41 and 35-45 mmHg respectively). Mechanical Ventilation of Mice 37

Table 4-1 Reported variables of spontaneous reclpiration of adolescent and adult mice. If bodvweight was unknown (*), then Ve is expressed in [mlog-lomin-l] else as [mlomin-1] with the bodyweight given. Abbreviations: bw = bodyweight, RR = respiratory rate, Vt = tidal volume. TilT! = inspirato/y fraction, Ve ~ minute volume, Ref = reference, rsv = respiratory syncytial virus injection. AdaptedFomI

Mouse Bw RR Vt Ve Ref. Condition Ti/Tt Strain [g) [min-I] [ml] Irml'min-I] [No.] 129 Sv/J x ICR 37.9 Restrained 195 0.12 0.47 24.0 7 CF-l 25.0 Restrained 272 0.12 (?) 32.6 10 C3H/HeJ 23.5 Unrestr. 177 0.24 0.32 24.7 3 AKRlJ 29.7 Unrestr. 222 0.29 0.29 37.1 3 129/J 18.1 Unrcstr. 230 0.17 0.29 23.2 3 DBAl2.1 25.1 Unrestr. 237 0.19 0.37 26.6 3 BALB/c.l 22.1 UnrestI'. 241 0.17 0.31 23.9 3 AIJ 22.9 Unrestr. 247 0.19 0.29 28.9 3 SJLlJ 22.3 Unrestr. 266 0.14 0.33 23.2 3 C57BLl6J 23.4 UnrestI'. 272 0.16 0.29 25.5 3 Wild type 30.0 (7) 180 0.15 (7) 27.0 11 BALB/c 22-26 Restrained 307 (7) 0.47 (7) 2 BALB/c 22-26 Restr.+rsv 420 (7) 0.33 1(7) 2 C57BLl6 (7) Unrestr. 210 (7) (7) (7) 12 Ovcrex.Gsa (7) Unrestr. 269 (7) (7) (?) 12 Wild type (7) (7) (") (7) (7) 1.1-1.3* 13 IeR pregnant 46 (7) (7) (7) II?) 3.5* 14 IeR non-pregn. 35 (7) (7) en (7) 3.0* 12

MURINE MECHANICAL VENTILATION

The mechanical ventilation of mice is required under numerous circumstances, the most important probably being an anesthesiological indication. A major goals of anesthesia is achieve more controlled experimental conditions, including the inhibition of spontaneous movements and stable cardiovascular parameters. However, it is important to realize that most potent sedative, anesthetic or analgesic drugs used in mice cause depression of spontaneous ventilation, e.g. RR and Vt. Although these drugs might decrease ventilatory requirements by a reduction of metabolism, this usually does not compensate for the depression of spontaneous ventilation. This mismatch of metabolism and ventilation may lead to hypoxia, hypercapnia, acidosis and consecutive disturbances of the homeostasis. This has been confirmed in CD-l mice (anesthesia with 350 mg'kg-1 chloralhydrate and xylazine 4 mg' kg-l i.p.), where spontaneously breathing mice became hypoxic (Pa02 :::: 60 mmHg), hypercapnic (PaC02 :::: 62 38 Physiological Genomics of the Critically III Mouse

mmHg) and acidotic (pH = 7.2), whereas values for the mechanically ventilated mice where markedly more physiological (Pa02 = 82 mmHg, PaC02 = 43, pH = 7.3) 20. Therefore a monitored mechanical ventilation supports the maintenance of a physiological relevant, stable murine model. 21 The control of ventilatory parameters is especially important in research using genetically altered mice. These are often more sensitive to standard laboratory procedures l9 and/or have specific alterations in their control of breathing. Typical examples of this are the obese leptin-deficient mice, showing respiratory depression already in the awake stateI9,22, mice lacking the brain derived neurotropic factor23 , RET protooncogene deficient mice with depressed ventilatory responses, and endothelin-l deficient mice with altered blood gasvalues (e.g. significantly lower P02 than wild type) and impaired respiratory response to hypoxia and hypercapnia7. However, it is often unknown at which stageduring development and under which conditions alterations occur in the respiratory system or the control of breathing6,24. Even among inbred wild type strains the control of respiration differs substantially, e.g. the C57BLl6strain was classified as high-responsive to hypercapnia, whereas the DBA/2strain as high-responsive to hypoxia3. Recently, comparing C3H1HeJ and C57BLl6 strains, differences in the respiratory response have been characterized for hypoxic25 and hypercapnic26 stimulation. In the latter study, hypercapnia induced in C3H1HeJ-mice a slow, but deep breathing pattern, whereas C57BLl6 mice showed a rapid, shallow breathing. Therefore, in these animals mechanical ventilation offers the possibility to study other effects of differences in genetic background independent of changes in ventilation. Certain surgical procedures have an improved outcome or can only be performed with support of mechanical ventilation. Examples are the bilateral ligation of the carotid arterio, where non-ventilated mice often die from respiratory disturbances, or the growing field of cardiac research that needs an open-chest murine model27-30. Hereby opening of the thorax and the adherent interpleural space induces lung collapse with respiratory insufficiency, whereas with support of a mechanical respirator the lungs can be inflated and ventilated.

AIRWAY ACCESS IN MICE

Several options exist to access and maintain the airway in mice. In early studies using a mechanical ventilator with active inspiration and expiration for mice3 \ the airway was accessed by advancing a tube through the mouth Mechanical Ventilation ofMice 39 into the pharynx, but not further into the trachea. Although technically less demanding than endotracheal intubation, this attempt risked gastric gasinsufflation, including aspiration of gastric content. A different, non• traumatizing mode to access the airways was performed with the Brady• Newsom apparatus32 : It consists of a gaschamber around the murine head, sealed from the thorax by a tight neoprenecollar. Elevating the pressure in this chamber leads to increased airway pressures, with respect to ambient air. Thus, this concept provides continuous positive airway pressure (CPAP) if the mouse ventilates spontaneously, or mechanical ventilation if periodic pressure changes in the chamber are generated. Although this apparatus has advantages, e.g. the technically less demanding airway access, it is also hampered by the disadvantage of gastric gas insufflation and aspiration of gastric content. To circumvent this problem, mechanical ventilation of mice today is usually performed after securing the airways by orotracheal intubation or via tracheotomy and consecutive endotracheal intubation. Tracheotomy has been performed in preterm neonate mice (I .15 g bodyweight) with insertion of polyethylene tubes (SP8, Natsume, Tokyo, Japan; tapered 0.4 mm outer diameter (OD»7, whereas for adult mice the OD of the endotracheal tube ranges from 0.9 mm (25-38 g mice) 29 to 1.1 mm (20-35 g)mice7 For experiments requiring recovery after surgery, temporary orotracheal intubation is performed, ideally via direct laryngoscopy with a purpose• made laryngoscope! I. The visualization of relevant airway structures requires a strong small-focus lightsource, allowing transillumination of the laryngeal cavity (with vocal cords and tracheal orifice), when put on the ventral cervical region from outside. Orotracheal intubation is performed with or without visual confirmation of the tube's tipposition (tubeposition judged by respiratory excursions). Visualization of the trachea (after surgical access to the deeper cervical structures) and controlled advancement of the endotracheal tube allows more accurate positioning of the tube, but is more invasive and has therefore an increased morbidity and mortality. For animals sacrificed at the end of the experiment, direct surgical access to the airways may be the best option. Since most of our mouse-surgeries require preparation of deeper cervical structures (e.g. catherisation catheterisation of a carotid artery and jugular vein) performing tracheotomies is convenient, with the additional advantage of minimized dead space (Vd). A reliable surgical procedure for this airway access is briefly described: After induction of anesthesia the mouse is put into supine position and the head is reclined (silk or rubber band around the upper incision teeth and attached to the operating surface) to allow easy access to the ventral neck. A median cervical skin incision (upper thorax aperture to lower jaw) is performed and layerwise skin, fascia, fat and connective tissue 40 Physiological Genomics of the Critically III Mouse

covering the thyroid gland are removed. The thyroid lobes are divided bluntly at their isthmus, pulled aside and kept retracted by bUlldogclamps. The pretracheal muscles are spread bluntly and pulled aside to allow access to larynx and trachea. Tracheotomy is performed by a transversal cut between two tracheal rings, usually in the lower third of the trachea. For the endotracheal intubation vascular PEcatheters (Abbocath-T@, Abbott• Venisystems) are used, cut to a length of about 5 mm with an angled tip (45°) to allow a smooth introduction. The correct position of the endotracheal tube is confirmed by judging chestexcursions, e.g. if respiration is still visible and symmetric. The tube is secured by a ligation (silk 6-0) around the trachea. If maintenance of spontaneous ventilation is desired, the endotracheal tube is shortened to minimize Vd and airway resistance. For mechanical ventilation, initially applied RR and/or Vt should be sufficiently higher than spontaneous RR and Vt of the anesthetized mouse to decrease respiratory drive (e.g. to prevent a "breathing against the ventilator") by moderate hyperventilation. After connecting the endotracheal tube, anesthesia can be safely deepened for an additional depression of ventilatory drive and facilitation of mechanical ventilation. Alternatively, this goal can be achieved (avoiding additional cardiocirculatory depression) by a non• depolarizing muscle relaxant (vecuroniumbromide 0.25 mg·kg- I bodyweight i.p.). Using this option it should be realized, however, that anesthesia depth cannot be judged from parameters such as spontaneous movement or limbwithdrawal to paw pinch, but instead to changes in haemodynamics.

MECHANICAL VENTILATION OF MICE

In general, there are two basic modes of respiration: spontaneous ventilation and controlled mechanical ventilation (CMV) where work of breathing is taken over by the ventilator. Inbetween there is a variety of mixed forms, such asrespiratory assistance, augmenting inspiration or supported spontaneous ventilation with continuous positive airway pressure (CPAP), adapted for mice by Brady and colleagues32. However, these mixed modes are not largely established for mice yet, and will therefore not be discussed in detail. Although mechanical ventilation of mice has major advantages, it is confounded by difficulties: Obviously, the small size of the animals makes airway access more challenging than in other laboratory animals. For example, in 25-30 g mice, the trachea has an accessible length of only 3-5 mm and a circumference of about 2.5 mm. Appropriate endotracheal tubes have an outer diameter ranging from 0.4 mm for neonate mice7 to 1.0 mm for adult mice". The actual mechanical ventilation of the mouse is Mechanical Ventilation ofMice 41 complicated by the technically demanding respiratory pattern of this species, e.g. the high RR, the low TilTt ratio and the small Vt, the latter also demanding a minimized dead space (V d) to prevent rebreathing. In commercially available rodent ventilators, the relatively large system V d allows substantial gas compression, thereby minimizing the Vt delivered to the mouse. Ewart et al. 17 reported that 2 ml of added Vd (compliance ~ 0.002 ml/cm H20) and a peak inspiratory pressure of 40 cm H20 reduce the delivered Vt by about 50%. The most obvious difference between spontaneous respiration and usual modes of mechanical ventilation are inverted pressure relations, with respect to ambient pressure, during the respiratory cycle: During spontaneous inspiration the expansion of the intrathoracic volume (mainly caused by contraction of the diaphragm and extension of the rib cage) generates a negative intrathoracic pressure and allows gas flow into the lungs. During expiration mainly passive elastic forces of lung and rib cage generate a positive intrathoracic pressure, leading to exhalation of air. Applying usual mechanical ventilation, the air is pushed during inspiration into the mice's lungs, leading to a positive intrathoracic pressure. During expiration intrathoracic pressure equilibrates with ambient pressure (intermittent positive pressure ventilation, IPPV) or remains positive (continuous positive pressure ventilation, CPPV). Therefore it is important to note that mean airway pressure during mechanical ventilation is higher than during spontaneous ventilation. These higher pressures contribute to side effects induced by mechanical ventilation. 42 Physiological Genomics ofthe Critically III Mouse

Table 4-2. Parameters of mechanical ventilation in murine closed and open-chest experiments. If bodyweight (bw) was unknown, the age in weeks [wk] is given as surrogate. Vt is expressed in [ml], only if bodyweight was unknown, the Vt is expressed in [LJlog-l]. Abbreviations: bw = bodyweight, RR = respiratory rate, Vt = tidal volume, Ti/Tt = inspiratory fraction, P = PEEPpressure [cm H20], Ref. = reference, CG = Capnography, OC = open chest. Adapted from I.

Mouse bw Venti- RR Vt TilTt Extra Ref. Strain [g] Lator ['min-I] [ml] [No.] C57 SV 7-8wk Harvard 150 5-6 ,J.J.g- I (?) P 3-4 34 129 nNOS(-I-) 7-8wk Harvard 150 5-6 1lJ.g-I (?) P 3-4 34 ICR 35-42 Harvard 150 0.28-0.34 (?) P~3 15 AlJ 21.8 Custom 120 0.17-0.2 (?) (?) 17 SV-129 19-31 CYE 120-170 0.35-0.45 0.14-0.28 CG 7 ICR 35-46 CYE 140 0.7 0.47 (?) 14 C3HIHe 23.3 Custom 120 0.17-0.2 (?) (?) 17 AlJ 15wk Custom 60 4-7 1lJ.g-I (?) (?) 35 BALB/c 15wk Custom 60 4-7 1l1'g-1 (?) (?) 35 C57BLl6 15wk Custom 60 4-7 1l1'g- 1 (?) (?) 35 C3H1He 15wk Custom 60 4-7 Ill'g-I m m 35 NIHMFI 12-28 (?) 168 (?) 0.21 P~2 36 129 38 Sinano 100-150 0.2-0.4 (?) CG 7 SYxICR ET-l (+1-) 38.4 Sinano 100-150 0.2-0.4 (?) CG 7 BALB/c 22-30 (?) 90 0.2 (?) cn 33 CD-I 35-40 Harvard 120 0.5 m (?) 20 BALB/c Young (?) 40 (?) 0.33 P~O 16 FVB 25-38 Harvard 110 0.2-0.4 (?) OC 29 ICR 33 Harvard 105 2.1-2.5 (?) OC 28 BALB/c 19-27 Harvard 90 0.8 (?) OC 30 Aicl C3H/HeJ 20-35 Custom 120 0.2 (?) OC 27

Mechanical ventilators are classified according to their method of cycling between inspiratory and expiratory phase. Small animal ventilators are usually time-cycled: The ventilator switches between the respiratory phases after a certain time has elapsed, depending on the preset respiratory rate. Although other methods of cycling might be favorable under certain experimental conditions (e.g. volume-cycled to prevent pulmonary volutrauma in murine open chest preparations) they are not yet well established. However, although the method of cycling is classified according to a single parameter (e.g. time), some mouse respirators allow a more sophisticated control of the ventilation by limiting relevant parameters: To prevent high inspiratory peak airway pressures, for example, most mouse ventilators allow the limitation of the ventilatory system pressure. Most Mechanical Ventilation ofMice 43 commercial mouse ventilators are time-cycled and pressure-limited, such as the mouse ventilator developed by our departmene7, FigA-l.

I ••n_~ w I

Figure 4-1. Principle of the mechanical mouse ventilator

The mouse ventilator developed in our departmene7 is based on the principle of the electric thumb: Inspiratory gas (from a conventional gas• mixing source) is directed via the fresh gas inlet (Fig. 4-1 , left, white arrows) into the inspiratory limb of the ventilator and further through the endotracheal tube into the mouse (Fig. 4-1, bottom). To initiate expiration (black arrows), an electromagnetically driven flap, covering the gas outlet of the ventilator (Fig. 4-1, right) during inspiration, is rapidly removed. This electromagenetic valve, i.e. the electricthumb, is operated by a programmable controller (not shown). The inspiratory and expiratory pressures (and flows) are separately regulated by resistors in the inspiratory• and expiratory limb of the ventilator (Fig. 4-1, black wedges). Primary goal of mechanical ventilation is delivery of an adequate respiratory minute volume (Ve) to the murine lung. To achieve this, the RR and Vt have to be adjusted to supply the actual ventilatory requirements. RR in most current mouse ventilators is the preset cycling parameter, but Vt• lung may vary, depending on the compliance of lungs and tubing and whether respiration is pressure-limited. A decrease in compliance of the respiratory system, for example in murine models of interstitial pulmonary diseases (ARDS, pneumonia), increases the airway pressure for a given Vt ventilator. If ventilation is pressure-limited, then Vt Illng will be considerable less than Vt ventilator. In contrast, if higher peak pressures are accepted to maintain Vt 11Ing, the risk to traumatize the delicate murine lung will increase38 • To prevent occurrence of these high pressures, inspiration time relative to expiration can be increased for a given Vt (up to inverse ratio ventilation (lRV), with inspiration longer than expiration). Disadvantages of a higher 44 Physiological Genomics of the Critically III Mouse

inspiratory fraction during mechanical ventilation are possibly insufficient times for complete exhalation ("air trapping") and the prolonged phases of elevated intrathoracic pressure, which may cause a decreased cardiac preload and consequently lowered blood pressures21 • A feature provided by some mouse ventilators is the possible application of external positive end-expiratory airway pressure (PEEP). During expiration, airway pressure is not allowed to equilibrate with ambient pressure (0 cm H20), but kept at a higher level (usually 2-10 cm H20). Mechanical ventilation with PEEP prevents the endexpiratory collapse of small airways and possibly recruits atelectatic pulmonary areas for gas• exchange by increase of functional residual capacity (FRC). Since pulmonary compliance increases (up to a certain degree) with increased FRC, ventilation with PEEP supports pulmonary inflation. Whether this holds true for mice has not yet been demonstrated. Additionally, ventilation with PEEP might mimic the physiologic end-expiratory tonus of inspiratory muscles and glottis of spontaneous ventilating mice. Typical side effects, demonstrated in a murine model of PEEP, are discussed below. A list of ventilator settings is given in 4-2. Most experiments have mechanical ventilation set by RR = 100-150·min-\ Vt = 0.2-0.7 ml , Ti/Tt 0.2-0.4. When these values are compared to values of spontaneous ventilating mice (Table 4-1), it becomes obvious that lower RR and higher Vt are commonly used to mechanically ventilate mice. Intermittent positive pressure ventilation (IPPV) and continuous positive pressure ventilation (CPPV) are the two common modes in murine mechanical ventilation, as provided by commercial ventilators. However, for the ventilation of newborn mice (1.0-1.5 g bodyweight, Vt 5-20 /-11) the concept of the "iron lung", based on the principle of body surface negative pressure ventilation has been adopted6 to prolong survival-time of NMDA• receptor mutant mice39, usually dying within 20 h after birth Only a few studies using mechanical ventilation in mice report on additional ventilatory parameters, like inspiratory gas flow or pressures in the ventilatory system. Using a RR of 140·min-l , a Vt of 0.7 ml and an inspiratory time of 0.2 sec (resulting in an inspiratory fraction (Ti/Tt) of 0.4) Furukawa et al. 14 achieved with inspiratory flows of 200 ml·min- I an end- . . f 35 InSpIratory pressure 0 8-10 cm H20. Yasue et at. ventilated mice of different strains with a rather low RR of 60·min-l , a Vt of 7-10 ml·kg- I and maintained ventilatory pressures below 5 cm H20. In general, it is reported that normal airflow pressures for mice should be in the range from 5-10 cm H20 40, although direct evidence for this range is lacking. Data are also available on ventilationregimen for murine open-chest preparations: Guo et al. 28 compared respiratory rates of95, 105 and 121·min• I (room air with oxygen (21·min- I ), Vt = 2.2 ml with endotracheal tube Mechanical Ventilation ofMice 45 loosely connected) in male ICR mice (8-12 weeks old, mean body weight 33.3 g) with respect to the resulting arterial blood gases. Adequate ventilation was obtained with a RR of 10S'min-I, resulting in an arterial Pa02 of 327 mmHg (177 and 321 mmHg for 9S'min-! and 121'min-! respectively), an arterial PaC02 of31 mmHg (43 and 18 mmHg for 9S'min-! and 121'min-! respectively) and an arterial pH of 7.4 (7.3 and 7.S for 9S'min• ! and 121'min-! respectively). Other regimens for the ventilation of open• chest mice are listed in table 4-2.

MONITORING OF MECHANICAL VENTILATION

During controlled mechanical ventilation (CMV), the most frequently applied mode of artificial respiration in mice, the work of breathing is taken over by the respirator. To facilitate this controlled mechanical ventilation (e.g. prevention of "breathing against the ventilator") it is usually performed under deep anesthesia. This implies, that relevant compensatory mechanisms (e.g. onset of spontaneous ventilation induced by hypercapnia or hypoxia) are impaired and underline the importance of monitoring mechanical ventilation. The attempt to monitor mechanical mouse ventilation is complicated by the fact that even standard variables for ventilation are not yet established, or show conflicting results (see Table 4-1). Several possibilities are available to monitor adequacy of ventilation. A highly subjective way, requIrIng experience, is to observe the thoraxexcursions with respect to rate, amplitude, symmetry and regularity as indicators of ventilatory rate, tidal volume, endotracheal tube position and attempt of spontaneous ventilatory effort. Dalkara et al. 2! suggest for a physiological mouse ventilation (mean PaC02 = 3S mmHg) to "keep the volume at a level at which thoracic movements first become noticeably observed". Information on the oxygenation can be obtained from the ear and tail skin colour, e.g. with cyanosis indicating poor oxygenation. These measures might serve as a first orientation in the evaluation of the adequacy of mechanical ventilation. More objective and in-depth measures are airway pressures and partial pressures. Airway pressures: The direct, non-invasive measurement of murine airway pressures is technically difficult, therefore substitutes have been established to obtain estimates. In commercially available mouse ventilators, the airway pressures are measured inside the ventilator. The advantage of this construction is that it circumvents the need for an additional pressure transducing line, ideally originating from the endotracheal tube itself, to a pressure transducer. A major disadvantage, however, is that pressures measured in the ventilator need to be extrapolated to airway-pressures within 46 Physiological Genomics ofthe Critically III Mouse

the endotracheal tube. This extrapolation is determined by the compliance of the respiratory gases and the tubing system ("signal damping"). It is therefore desirable to measure airway pressures as close as possible to the endotracheal tube. To this end we have developed an integrated endotracheal tube within a small aluminum frame (lOx5x2 mm, 5 g), consisting of temperature controlled adapters for inspiratory and expiratory tubing, an inlinecapnography cell (V d = 25 Ill) and a liquid-filled airway-pressure transducer. Although airwaypressures are usually monitored to maintain safe values for mean and peakairway pressure, this measure can also serve to titrate damage in murine models of respirator induced lung impairment, such as baro- or volutrauma38, where tidal volumes (Vt) have been adjusted to achieve traumatizing target airway pressures. Partial gas pressures: Inflow freshgas composition to the mechanical ventilator can be monitored with respect to the concentrations of the different respiratory gases, e.g. oxygen, nitrous oxide and volatile anesthetics. When rebreathing of exhaled air is excluded in the ventilatory circuit, analysis of fresh gas streaming to the respirator should accurately reflect the composition of inspiratory gases. This attempt is advantageous, compared to sample-lines attached to the inspiratory tubing-limb of the system, because it circumvents the need for gas extraction from the inspiratory tubing with the consequence of decreased inspiratory airway• pressures and ventilatory tidal volumes. The actual measurement can be performed online with commercially available multi-gas analyzers. It has been argued that ventilation with room air may not be ideal in mice40, based on observed high murine P50-values (P02 corresponding to 50% hemoglobin-saturation) of 65 mmHg41. However, most studies have found a P50 ~ 40 mmHg for the mouse, when measured under standard conditions (pH = 7.4 , PC02 = 40 mmHg, 37oC)18,42-44. A P50 of ~ 40 mmHg is still markedly right-shifted when compared to humans (P50 = 25-30 mmHg). Nevertheless, although even with a high P50 ~ 65 mmHg blood is virtually 100% saturated at room air41 , increased Fi02 may be helpful in the mouse when uncertainty exists as to whether mechanical ventilation optimally expands the lungs. Capnography: In contrast to maintenance of sufficient arterial oxygenation, which even under conditions of poor ventilatory support can be achieved by an increased fraction of inspiratory oxygen (Fi02), adequate pulmonary elimination of CO2 is the more challenging aspect of mechanical ventilation. Although the arterial blood gas analysis is regarded as the gold standard for evaluation of proper ventilation, the drawbacks are that it is a discontinuous and invasive method requiring a substantial volume of arterial blood i.e.O.l-0.2 ml for a single measurement, 5-10% of the total blood volume of a 25 g mouse, the latter aspect adding new sources of instability Mechanical Ventilation ofMice 47 to the model. Though often neglected, measurement of respiratory carbon dioxide concentration (capnography) is therefore recommended in mouse research. Especially the determination of the end-tidal CO2 (etC02), as measure for alveolar C02concentrations, is described as essential to maintain stable physiological parameters during mechanical ventilation of mice21 • However, capnography has rarely been performed in mice due to technical difficulties, such as gas dilution artifacts occurring inside stream systems which prevent accurate detection of end-tidal CO2 peaks or plateaus. Therefore, to use the potential of capnography to its full extent, fast responding mainstreamcapnography is required. To this end, we have developed an integrated mouse endotracheal tube with an implemented capnographycell with minimal gas-mixing artifacts. Since this cell is located in the endotracheal tube it allows real-time inspiratory and expiratory mainstream breath-by-breath capnography Figure 4-2 presents a typical capnography curve using this devic, , showing several single breath capnography curves (inner section with high chart speed, RR - 100 min-I).

Figure 4-2. Capnography (C02) and mean arterial pressure (MAP) during mechanical ventilation of an anesthetised mouse (CS7BL/6, 23 g). Note the correspondence of respiratory phase and haemodynamic effect (black arrow = end inspiratory, white arrow = end expiratory). Adapted from I and 37. 48 Physiological Genomics ofthe Critically III Mouse

Figure 4-3. Effect of blood withdrawal (0.2 ml, black arrow) and immediate resuscitation with albumin solution (0.2 ml, white arrow) on capnography. For details see text. Adapted from J and 37.

Capnography IS not only a measure ofthe adequacy of mechanical ventilation, but can also serve to detect changes in haemodynamics. An example for this is given in Figure 4-3: During steady state anesthesia 0.2 ml blood was withdrawn from an arterial line (black arrow). The end-tidal carbon dioxide concentration drops immediately, likely due to a decrease in pulmonary perfusion (decreased cardiac output). Resuscitation is performed by rapid infusion of 0.2 ml albumin solution (white arrow), that increases capnography values back to baseline, probably by restoring pulmonary perfusion. Since time resolution is enhanced by our breath-by-breath capnography, the relation between expiratory CO2 and specific events can be described precisely. Additionally, the capnography curves can be analyzed with respect to irregularity (e.g. due to intermittent spontaneous breathing) and shapeaIterations (e.g. airway obstruction).

CONCLUSION

In conclusion, control of mouse respiration is a field of expanding interest. Although spontaneous ventilation is the more physiological mode of breathing, only mechanical ventilation maintains physiological values in situations with impaired spontaneous breathing, as occurring during anesthesia, surgery or constitutively in murine strains with impaired spontaneous ventilation. There are currently several commercially available small rodent ventilators feasible for use in mice. If mechanical ventilation is performed, then monitoring of this intervention is required, ideally with means of capnography. Research has to be performed on how the most Mechanical Ventilation ofMice 49

physiological ventilation can be achieved in mice, which ventilatory patterns are appropriate and how ventilation related side effects can be limited.

References

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